Figure 4. GAPDH Reaction.

The reaction catalyzed by GAPDH involves two processes: Oxidation of the glyceraldehyde-3-phosphate (G3P) aldehyde to a carboxylic acid by NAD⁺, and joining of the G3P carboxylic acid and the NAD⁺ orthophosphate to form 1,3-bisphosphoglycerate (BPG). The first step of this mechanism involves nucleophilic attack by the Cys-149 (human analog Cys-152) SH-group on the carbonyl of G3P, resulting in formation of a hemithioacetal. Acting as an acid catalyst, His-176 (human analog His-179) facilitates hemiacetal development by forming an ion pair with the Cys-149 SH-group, enhancing Cys-149 reactivity, and favoring the nucleophilic attack of GAPDH by the thiolate [76]. A hydride transfer to NAD⁺ oxidizes the hemiacetal intermediate to a high energy thioester, facilitated by His-176, now acting as a base catalyst during the oxidation step [75, 76]. His-176 stabilizes the hemithioacetal intermediate by hydrogen bonding the hemiacetal hydroxyl group to the N^ε of its imidazole ring, while the acylenzyme intermediate, is stabilized by hydrogen bonding with the protonated imidazole N^ε of His-176 [76]. Finally, the thioester undergoes nucleophilic attack by an inorganic phosphate (P_i), facilitated again by His-176 acting as an acid catalyst, to form BPG [75–77], which is then released from the ternary complex, along with NADH (adapted from [76]).